Bio-Rad Rat Diabetes Assays - Manual (Page 42)

40
Appendix: Protease Inhibitors
Refer to the recommended protocol below for preparing the inhibitors.
Materials
n
DDP-IV Inhibitor (Sigma Aldrich, K4264, Molecular Weight: 370.24):
Store at 4ºC
n
Aprotinin (Sigma Aldrich, A3428, 3–8 TIU/mg or 3,900–10,400 KIU/mg):
Store at 4ºC
Preparation of Protease Inhibitors Stock Solution
1. Preparation of 10 mM DDP-IV inhibitor solution
a) Weigh 18.5 mg of DDP-IV inhibitor.
b) Dissolve completely in 5 ml of 0.9% NaCl.
c) Aliquot and store at –20ºC. Avoid repeated freeze/thaw cycles.
2. Preparation of 1.3% Aprotinin
a) Weigh 100 mg Aprotinin.
b) Dissolve completely in 7.5 ml of 0.9% NaCl.
c) Aliquot and store at –20ºC. Avoid repeated freeze/thaw cycles.
Blood Sample Preparation
1. Collect whole blood in plasma collection tubes.
2. Add 10 μl of 10 mM DDP-IV inhibitor per 1 ml of whole blood for a
final concentration of 100 μM.
3. Add 10 μl of 1.3% Aprotinin per 1 ml of whole blood for a final
amount of >500 KIU per 1 ml of whole blood.
4. Invert the tubes several times to mix the protease inhibitors with blood.